Brianna Dalesandro

Nature Chemical Biology, Published online: 15 December 2022; doi:10.1038/s41589-022-01194-1

This article presents a synthetic genetic program for orthogonal, tunable and programmable control of bacterial lifestyle and associated phase-specific gene expression, offering a versatile platform for microbial engineering in complex contexts.

The bridging pattern of the lantibiotic pseudomycoicidin, a (methyl-)lanthionine-containing antibiotic peptide that is produced by Bacillus pseudomycoides is elucidated here. An alanine scan and MALDI TOF MS of variant peptides showed that the peptide is characterized by a compact structure with two double ring systems, one disulfide bridge, and a possible conserved lipid II binding motif.

Abstract

Lantibiotics are post-translationally modified antibiotic peptides with lanthionine thioether bridges that represent potential alternatives to conventional antibiotics. The lantibiotic pseudomycoicidin is produced by Bacillus pseudomycoides DSM 12442 and is effective against many Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. While prior work demonstrated that pseudomycoicidin possesses one disulfide bridge and four thioether bridges, the ring topology has so far remained unclear. Here, we analyzed several pseudomycoicidin analogues that are affected in ring formation via MALDI-TOF-MS and tandem mass spectrometry with regard to their dehydration and fragmentation patterns, respectively. As a result, we propose a bridging pattern involving Thr8 and Cys13, Thr10 and Cys16, Ser18 and Cys21, and Ser20 and Cys26, thus, forming two double ring systems. Additionally, we localized the disulfide bridge to connect Cys3 and Cys7 and, therefore, fully elucidated the bridging pattern of pseudomycoicidin.

A heterodimeric macrocyclic peptide inhibitor against VEGF-VEGFR2 interaction was designed and produced by a microbial secretion system. It showed stronger binding potency and >1000 higher inhibitory activity against VEGFR2 than parental monomeric macrocyclic peptides.

Abstract

The inhibition of protein-protein interactions (PPIs) is an effective approach for therapy. Owing to their large binding surface areas to target proteins, macrocyclic peptides are suitable molecules for PPI inhibition. In this study, we developed single-chain tandem macrocyclic peptides (STaMPtides) that inhibits the vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2). They were artificially designed to comprise two different VEGFR2-binding macrocyclic peptides linked in tandem by peptide linkers and secreted by Corynebacterium glutamicum. Most potent VEGFR2-inhibitory STaMPtides with length-optimized linkers exhibited >1000 times stronger inhibitory activity than their parental monomeric peptides, possibly due to the avidity effect of heterodimerization. Our approach of using STaMPtides for PPI inhibition may be used to inhibit other extracellular factors, such as growth factors and cytokines.

Glycopeptide antibiotic (GPA) crosslinking is performed by cytochrome P450 (Oxy) enzymes and is essential for antibiotic activity. We show that these Oxy enzymes are tolerant of extension at the N but not the C termini of their peptide substrates. Alterations in peptide length were also able to alter the order of activity observed for the Oxy enzymes.

Abstract

The glycopeptide antibiotics (GPAs) are a clinically approved class of antimicrobial agents that classically function through the inhibition of bacterial cell-wall biosynthesis by sequestration of the precursor lipid II. The oxidative crosslinking of the core peptide by cytochrome P450 (Oxy) enzymes during GPA biosynthesis is both essential to their function and the source of their synthetic challenge. Thus, understanding the activity and selectivity of these Oxy enzymes is of key importance for the future engineering of this important compound class. Recent reports of GPAs that display an alternative mode of action and a wider range of core peptide structures compared to classic lipid II-binding GPAs raises the question of the tolerance of Oxy enzymes for larger changes in their peptide substrates. In this work, we explore the ability of Oxy enzymes from the biosynthesis pathways of lipid II-binding GPAs to accept altered peptide substrates based on a vancomycin template. Our results show that Oxy enzymes are more tolerant of changes at the N terminus of their substrates, whilst C-terminal extension of the peptide substrates is deleterious to the activity of all Oxy enzymes. Thus, future studies should prioritise the study of Oxy enzymes from atypical GPA biosynthesis pathways bearing C-terminal peptide extension to increase the substrate scope of these important cyclisation enzymes.

Peptides provide safe, potentially potent, and chemically diverse prospects in the development of antiviral therapeutics. As viruses continue to evolve, new antiviral drugs are intrinsic to preventing future global pandemics. Peptides discovered from natural sources, as well as those laboratory-developed and modified with other biomolecules, have shown promising results in mitigating viral infections.

Abstract

Peptides are ideal candidates for the development of antiviral therapeutics due to their specificity, chemical diversity and potential for highly potent, safe, molecular interventions. By restricting conformational freedom and flexibility, cyclic peptides frequently increase peptide stability. Viral targets are often very challenging as their evasive strategies for infectivity can preclude standard therapies. In recent years, several peptides from natural sources mitigated an array of viral infections. In parallel, short peptides derived from key viral proteins, modified with chemical groups such as lipids and cell-penetrating sequences, led to highly effective antiviral inhibitor designs. These strategies have been further developed during the recent COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2. Several anti-SARS-CoV-2 peptides are gaining ground in pre-clinical development. Overall, peptides are strong contenders for lead compounds against many life-threatening viruses and may prove to be the key to future efforts revealing viral mechanisms of action and alleviating their effects.

Clostridioides difficile cells are sensitive to vancomycin, which targets the D-Ala-D-Ala moiety of peptidoglycan and serves as a first-line drug for treating C. difficile infection. High, unregulated expression of the VanG protein, which synthesizes D-Ala-D-Ser, allowed D-Ala-D-Ser incorporation into peptidoglycan and conferred resistance to vancomycin but only at a low level. Growth in the presence of D-Ala-D-Ser of cells that are unable to synthesize D-Ala-D-Ala did not increase further the cells' vancomycin resistance.

Abstract

A Clostridioides difficile strain deficient in the ddl gene is unable to synthesize the dipeptide D-Ala-D-Ala, an essential component of peptidoglycan and the target of vancomycin. We isolated spontaneous suppressors of a ∆ddl mutation that allowed cell growth in the absence of D-Ala-D-Ala. The mutations caused constitutive or partly constitutive expression of the vancomycin-inducible vanG operon responsible for the synthesis of D-Ala-D-Ser, which can replace D-Ala-D-Ala in peptidoglycan. The mutations mapped to the vanS or vanR genes, which regulate expression of the vanG operon. The constitutive level of vanG expression was about 10-fold above that obtained by vancomycin induction. The incorporation of D-Ala-D-Ser into peptidoglycan due to high expression of the vanG operon conferred only low-level resistance to vancomycin, but VanG was found to synthesize D-Ala-D-Ala in addition to D-Ala-D-Ser. However, the same, low resistance to vancomycin was also observed in cells completely unable to synthesize D-Ala-D-Ala and grown in the presence of D-Ala-D-Ser. D-Ala-D-Ala presence was required for efficient vancomycin induction of the vanG operon showing that vancomycin is not by itself able to activate VanS. D-Ala-D-Ser, similar to D-Ala-D-Ala, served as an anti-activator of DdlR, the positive regulator of the ddl gene, thereby coupling vanG and ddl expression.

A single-atom (O→S) substitution to introduce a backbone thioamide linkage in a peptide facilitates a Ag^I-promoted, site-specific generation of Asn N-glycopeptides.

Abstract

A site-specific method for the preparation of N-glycosylated peptides is described. Incorporation of a peptide backbone thioamide linkage adjacent to an Asp residue facilitates a Ag^I-promoted, site-specific conversion to N-glycosylated Asn residues in peptides.